Metabolism

Metabolic Stability – Liver Microsomes

This assay is used to determine the percent remaining of a test compound incubated with human, rat, dog, primate or mouse liver microsomes in the presence of NADPH Catalog number.

Required from Customer

• Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
• Molecular mass (exact mass) of test compound and its salt form
• MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

• Table of % remaining of test compound at each time point
• Calculated t_1/2 of test compound and control
• Calculated in vitro intrinsic clearance of test compound

Substrate

• Test compound at 1 µM dissolved in DMSO, methanol, or acetonitrile

Assay System

• Pooled liver microsomes (≥3 donors), with 1 mM NADPH
  • Human: mixed gender
  • Rat, dog, primate or mouse: male only
• LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to test compound or control divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

• The assay is run with a single incubation (N=1)
• Incubate test compound (1 µM) at 37°C in buffer containing 0.5 mg/mL microsomal protein
• Initiate the reaction by adding cofactors, sampling at 0, 10, 20, 30, and 60 minutes
• Incubate positive control (5 µM testosterone) in parallel, sampling at 0, 10, 30, and 60 minutes

Assay QC

• The control compound, testosterone, is run in a parallel assay to verify the activity of the CYPs
• After the final time point, fluorimetry is used to confirm the addition of NADPH to the reaction mixture
• T_1/2 of control must meet internal acceptance criteria

Notes

1. The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
2. This assay does not provide stability information of test compound in liver microsomes without NADPH.
3. Percent remaining at each time point is calculated from the peak area response ratio of test compound (or control) divided by the peak area response ratio of the 0-minute sample.
4. In vitro intrinsic clearance (CL_{int in-vitro}) = k/P (mL/min·mg protein), where k is the elimination rate constant (min^-1) and P is the protein concentration (mg/mL).

Express Plus Metabolic Stability In Liver S9 Fraction

This assay is used to determine the percent remaining and intrinsic clearance of a test compound incubated with liver S9 fraction in the presence of cofactors.

Required from Sponsor

• Either a minimum of 300 μL of test compound at 10 mM in DMSO, or 5 mg of powder
• Exact molecular mass of test compound and its salt form MSDS or handling and storage information (e.g., light sensitive, store at –20^oC, etc.)

Deliverables

• Table of % remaining of test compound at each time point
• Calculated t_1/2 of test compound and controls
• Calculated in vitro intrinsic clearance of test compound
• Notification if metabolites are detected

Substrate

• Test compound at 1 μM with final concentration of organic solvent < 0.25%

Assay System

• Pooled liver S9 fraction prepared with 1 mM NADPH, UDPGA, PAPS and GSH
• LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to test compound or control divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

• The assay is run with a single incubation (N=1)
• Incubate test compound at 37^oC in buffer containing 1.0 mg/mL S9 protein
• Initiate the reaction by adding cofactors, sampling at 0, 10, 20, 30, and 60 minutes
• Incubate positive controls (testosterone and 7-hydroxycoumarin) in parallel, sampling at 0, 10, 30, and 60 minutes

Assay QC

• The control compounds, testosterone and 7-hydroxycoumarin, are run in parallel to verify the enzymatic activity of the S9 fraction
• After the final time point, fluorimetry is used to confirm the addition of NADPH to the reaction mixture
• T_1/2 of control compounds must meet internal acceptance criteria

Notes

1. The results from this assay are sent to the customer in the Express Plus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
2. This assay does not provide stability information of test compound in liver S9 incubations without cofactors.
3. Percent remaining at each time point is calculated from the peak area response ratio of test compound (or control) divided by the peak area response ratio of the 0-minute sample
4. In vitro intrinsic clearance (CL_{int in vitro}) = k/P (mL/min·mg protein), where k is the elimination rate constant (min-1) and P is the protein concentration (mg/mL).
5. Relative abundance of the putative metabolites at each time point will be determined to provide kinetics of metabolite formation.

6. Up to 10 major putative metabolites will be identified and reported.

Options

1. Upon customer’s request, samples can be analyzed using an LTQ Orbitrap XL mass spectrometer
   • HRMS scan will be performed in an appropriate m/z range in order to detect all plausible metabolites
   • For an additional cost, the customer may request quatification of metabolites, if detected

Express Plus Metabolic Stability In Cyropreserved Hepatocytes

This assay is used to determine the percent remaining and intrinsic clearance of a test compound incubated with cryopreserved hepatocytes.

Required from Sponsor

• Either a minimum of 300 μL of test compound at 10 mM in DMSO, or 5 mg of powder
• Exact molecular mass of test compound and its salt form MSDS or handling and storage information (e.g., light sensitive, store at –20oC, etc.)

Deliverables

• Table of % remaining of test compound at each time point
• Calculated t_1/2 of test compound and testosterone
• Calculated in vitro intrinsic clearance of test compound
• Notification if metabolites are detected

Substrate

• Test compound at 1 μM with final concentration of organic solvent <0.25%

Assay System

• Pooled (at least 3 donors) cryopreserved hepatocytes
• LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to test compound or control divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

• The assay is run with a single incubation (N=1)
• Cell viability assessed by trypan blue exclusion prior to assay
• Incubate test compound at 37^oC in buffer containing 1.5 x 10⁶ hepatocytes/mL, sampling at 0, 15, 30, 60, and 120 minutes
• Incubate positive controls (testosterone and 7-hydroxycoumarin) in parallel, sampling at 0, 15, 30, 60, and 120 minutes

Assay QC

• The control compounds, testosterone and 7-hydroxycoumarin, are run in parallel to verify the enzymatic activity of the hepatocytes
• T_1/2 of testosterone elimination and rate of formation of 7-hydroxycoumarin conjugates must meet internal acceptance criteria

Notes

1. The results from this assay are sent to the customer in the Express Plus report format, which may include graphical representations of data and comparison with historical data for reference compounds.

2. This assay does not provide stability information of test compound in liver microsomes without NADPH.
3. Percent remaining at each time point is calculated from the peak area response ratio of test compound (or control) divided by the peak area response ratio of the 0-minute sample.
4. In vitro intrinsic cleance (CL_{int in vitro}) = k/P (mL/min·mg protein), where k is the elimination rate constant (min⁻¹) and P is the protein concentration (mg/mL).
5. Relative abundance of the putative metabolites at each time point will be determined to provide kinetics of metabolite formation.
6. Up to 10 major putative metabolites will be identified and reported.

Options

1. Upon customer’s request, samples can be analyzed using an LTQ Orbitrap XL mass spectrometer
   • HRMS scan will be performed in an appropriate m/z range in order to detect all plausible metabolites
   • For an additional cost, the customer may request quatification of metabolites, if detected

CYP IC₅₀ in Human Liver Microsomes – LC-MS/MS

This assay is used to determine the IC₅₀ for reversible inhibition of individual cytochrome P450 (CYP) enzymes by a test compound.

Required from Customer

• A minimum of 300 μL of test compound at 100 mM in DMSO, or 10 mg of powder
• Molecular mass (exact mass) of test compound and salt form
• MSDS or handling and storage information, e.g., light-sensitive, store at -20°C, etc.

Deliverables

• Percent of control activity of each CYP isoform in the presence of each concentration of test compound incubated under reversible conditions
• Percent inhibition of each CYP isoform by one concentration of a positive control inhibitor run in parallel under reversible conditions
• Microsomal lot certification for IC₅₀ of each CYP-specific positive control inhibitor under reversible conditions
• IC₅₀ of test compound for each CYP isoform

Substrate

• Seven concentrations of test compound dissolved in aqueous buffer, acetonitrile (≤1% final), methanol (≤1% final), or DMSO (≤0.1% final)

Assay System

• Pooled, mixed-gender, human liver microsomes from a minimum 10 donors with NADPH added in high molar excess
• Concentration of each individual CYP-specific probe substrate is near the K_m for the enzyme
• LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to a specific metabolite of each CYP-specific probe substrate divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

• The standard protein concentration is 0.25 mg/mL
• Run the assay with a single replicate (N=1 incubation)
• Include a maximum activity control, seven concentrations of test compound, and a single, high concentration of a positive control inhibitor
• Initiate reaction by adding NADPH
• Sample reaction mixture at a single time point (typically between 10 and 30 minutes)

Assay QC

• >50% inhibition by each positive control run in parallel

Notes

1. The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
2. IC50 values are estimated by fitting the experimental data (percent of control activity remaining at each concentration of test compound) to a sigmoidal model and non-linear regression analysis.

CYP Inhibition in Human Liver Microsomes – LC-MS/MS

This assay is used to screen for reversible inhibition of individual cytochrome P450 (CYP) enzymes by a single concentration of a test compound.

Required from Customer

• A minimum of 300 μL of test compound at 10 mM in DMSO, or 10 mg of powder
• Molecular mass (exact mass) of test compound and salt form
• MSDS or handling and storage information, e.g., light-sensitive, store at -20°C, etc.

Deliverables

• Percent inhibition of each CYP isoform by a single concentration of test compound under reversible conditions
• Percent inhibition of each CYP isoform by one concentration of a positive control inhibitor run in parallel under reversible conditions

Substrate

• The test compound at a single concentration (typically 10 μM) dissolved in aqueous buffer, acetonitrile (≤1% final), methanol (≤1% final), or DMSO (≤0.1% final)

Assay System

• Pooled, mixed-gender, human liver microsomes from a minimum 10 donors with NADPH added in high molar excess
• Concentration of each individual CYP-specific probe substrate is near the K_m for the enzyme
• LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to a specific metabolite of each CYP-specific probe substrate divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

• The standard protein concentration is 0.25 mg/mL
• Run the assay with a single replicate (N=2 incubation)
• Include a maximum activity control, a single concentration of test compound, and a single, high concentration of a positive control inhibitor
• Initiate reaction by adding NADPH
• Sample reaction mixture at a single time point (typically between 10 and 30 minutes)

Assay QC

• >50% inhibition by each positive control run in parallel

Notes

1. The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
2. Percent inhibition is calculated from the amount of metabolite formed in the presence of test compound relative to that formed in the absence of test compound, based on the peak area response ratio (PARR): % Inhibition = [(PARR without test compound – PARR with test compound) / (PARR without test compound)] x 100%.

CYP Phenotyping – Supersomes™

This assay is used to identify the CYP(s) involved in the metabolism of a test compound, through the use of CYP-specific Supersomes.

Required from Customer

• The test compound in powder form—a minimum of 10 mg
• Exact molecular mass of the test compound and its salt form
• Relevant solubility of test compound
• The MSDS or handling and storage information, e.g., light sensitive, store at -20ºC, etc.

Deliverables

• In vitro intrinsic clearance of test compound determined for each CYP, scaled to the relative abundance of each isoform
• Rank order of CYP isoform(s) responsible for test compound’s metabolism

Substrate

• Substrate Test compound at a single concentration (based on K_m; if not available, 1 µM will be used) dissolved in aqueous buffer, acetonitrile (≤1% final), methanol (≤1% final), or DMSO (≤0.1% final)

Assay System

• Individual CYP-specific Supersomes with NADPH
• Non-transfected control with NADPH run in parallel
• LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to the test compound divided by that of an analytical internal standard), without running a standard curve

Assay Conditions

• This assay is run with a single incubation (N=1) per treatment
• Incubate test compound at 37°C in the presence of CYP-specific Supersomes and non-transfected control
• Sample the reaction mixture at 0, 5, 10, 15 and 30 minutes

Batch QC

• Use fluorimetry to confirm the addition of NADPH to the reaction mixture

Notes

CYP Specific Substrates as Positive Controls

CYP Isoform	Probe Substrate	Metabolite
1A2	Phenacetin	Acetaminophen
2A6	Coumarin	7-OH coumarin
2B6	Bupropion	Hydroxyupropion
2C8	Amodiaquine	Desethylamodiaquine
2C9	Diclofenac	4’-OH diclofenac
2C19	S-mephenytoin	4’-OH mephenytoin
2D6	Bufuralol	1’-OH bufuralol
2E1	Chlorzoxazone	6-OH chlorzoxazone
3A4	Testosterone	6β-OH testosterone
3A4	Midazolam	1’-OH midazolam

CYP Phenotyping – Chemical Inhibitors

This assay is used to identify the CYP(s) involved in the metabolism of a test compound, through the use of human liver microsomes and CYP-specific chemical inhibitors.

Required from Customer

• The test compound in powder form—a minimum of 10 mg
• Exact molecular mass of the test compound and its salt form
• Relevant solubility of test compound
• The MSDS or handling and storage information, e.g., light sensitive, store at -20ºC, etc.

Deliverables

• In vitro intrinsic clearance of test compound determined for each CYP isoform
• Rank order of CYP isoform(s) responsible for test
• compound’s metabolism

Substrate

• Test compound at a single concentration (based on K_m; if not available, 1µM will be used) dissolved in aqueous buffer, acetonitrile (≤1% final), methanol (≤1% final), or DMSO (≤0.1% final)

Assay System

• Pooled, mixed-donor, human liver microsomes (minimum 50 donors) with and without NADPH
• CYP-specific chemical inhibitor(s)
• LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to the test compound divided by that of an analytical internal standard), without running a standard curve

Assay Conditions

• This assay is run with a single incubation (N=1) per treatment
• Incubate test compound at 37°C in the presence of human liver microsomes (0.5 mg protein/mL) and:
  • No NADPH (negative control)
  • NADPH (no-inhibitor control)
  • NADPH + a CYP-specific inhibitor (single concentration)
• Sample the reaction mixture at 0, 10, 20, 30 and 60 minutes

Batch QC

• Batch certification of human liver microsomes with CYP-specific probe substrates and CYP-specific chemical inhibitors
• Use fluorimetry to confirm the addition of NADPH to the reaction mixture

Options

1. The customer will be asked to specify: • the concentration(s) of the test compound • the CYP(s) to be screened
2. The customer can request: • additional replicates • additional or alternative time points • the use of a standard curve • that a positive control be run for each CYP

This assay is used to identify time-dependent inhibition of individual cytochrome P450 (CYP) enzymes by a test compound using the IC₅₀ shift approach.

Required from Customer

• A minimum of 25 mg of powder
• Exact molecular mass of test compound and its salt form
• Purity of test compound
• Metabolite standard(s) and purity (if available)
• MSDS or handling and storage information (e.g., light sensitive, store at -20°C, etc.)

Deliverables

• IC₅₀ of test compound and positive control vs. each CYP isoform under time-dependent inhibition conditions

Calculated IC₅₀ shift (-NADPH/ +NADPH)Substrate

• Test compound in aqueous buffer, acetonitrile or methanol (≤1% final)

Assay System

• Pooled, mixed-gender, human liver microsomes from a minimum 10 donors
• Concentration of each individual CYP-specific probe substrate is near the Km for that enzyme
• LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to metabolite of CYP-specific probe substrate) divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

• The standard protein concentration is 0.25 mg/mL
• Run the assay in duplicate (N=2 separate incubations)
• Include a maximum activity control, seven concentrations of test compound (typically serial dilutions from 100 µM), and seven concentrations of a positive control inhibitor
• After pre-incubating test compound with microsomes, with and without NADPH, at 37°C for 30 minutes, initiate enzyme activity assay by adding an enzyme-specific probe substrate (and NADPH, if not already present)
• Sample reaction mixture at a single time point consistent with the rate of formation of the metabolite
• Estimate IC₅₀ in the absence and presence of NADPH, calculate IC₅₀ shift (-NADPH/ +NADPH)

Assay QC

• IC₅₀ shift ≥3 for positive controls run in parallel

Notes

1. IC₅₀ values are estimated by fitting the experimental data (percent of control activity remaining at each concentration of test compound or positive control) to a sigmoidal model and non-linear regression analysis.

Individual CYP Substrates and Positive Control Inhibitors 

CYP Isoform	Probe Substrate	Metabolite	Positive Control Inhibitor
1A2	Phenacetin	Acetaminophen	Furafylline
2A6	Coumarin	7-OH coumarin	8-Methoxypsoralen
2B6	Bupropion	Hydroxybupropion	Thio-TEPA
2C8	Amodiaquine	Desethylamodiaquine	Gemfibrozil Glucuronide
2C9	Diclofenac	4’-OH diclofenac	Tienilic Acid
2C19	S-mephenytoin	4’-OH mephenytoin	Ticlopidine
2D6	Bufuralol	1’-OH bufuralol	Paroxetine
2E1	Chlorzoxazone	6-OH chlorzoxazone	Diethyldithiocarbamate
3A4	Testosterone	6β-OH testosterone	Troleandomycin
3A4	Midazolam	1’-OH midazolam	Troleandomycin

UGT Inhibition – Recombinant Enzymes and LC-MS/MS

This assay is used to assess the potential of a test compound to inhibit individual human UDP-glucuronosyltransferase (UGT) isoforms

Required from Customer

• Either a minimum of 600 µL of test compound at 10 mM in DMSO, or 10 mg of powder
• Exact molecular mass of test compound and its salt form
• MSDS or handling and storage information (e.g., light sensitive, store at -20°C, etc.)
• Stability of test compound in the presence of individual human UGTs
• Solubility of test compound in assay buffer

Deliverables

• Table of % inhibition of each UGT isoform by a single concentration of test compound
• Table of % inhibition of each UGT isoform by a positive control inhibitor

Substrate

• Test compound at a single concentration (typically between 10 and 100 µM, depending on solubility and customer input) dissolved in aqueous buffer, DMSO, acetonitrile, or methanol (final concentration of organic solvent ≤1%)

Assay System

• Individual human recombinant UGT isoforms, with 1 mM UDP-glucuronic acid (UDPGA) and alamethicin added
• Individual substrates in separate incubations
• LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to the glucuronide conjugate of each probe substrate divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

• The standard protein concentration is 0.25 mg/mL
• Run the assay in duplicate (N=2 separate incubations)
• Include a maximum activity control, a single concentration of test compound, and a single concentration of a positive control inhibitor
• Initiate reaction by adding 1 mM UDPGA
• Sample reaction mixture at a singe time point (typically between 30 and 60 minutes)

Assay QC

• Significant inhibition by each positive control run in parallel

Notes

1. The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
2. Percent inhibition is calculated from the amount of metabolite formed in the presence of test compound relative to that formed in the absence of test compound, based on the peak area response ratio (PARR): % Inhibition = [(PARR without test compound) – (PARR with test compound) / (PARR without test compound)] x 100%.
3. Pricing is based on screening for inhibition of at least five UGT isoforms per test compound.

UGT Isoforms, Probe Substrates and Metabolites

UGT	Probe Substrate	Conc. (µM)	Metabolite
UGT1A1, UGT1A3, UGT1A6, UGT1A9, UGT2B7	7-HFC	100	7-HFCG
UGT1A4	TFP	100	TFPG

Positive Control Inhibitors for Standard (hepatic) UGT Isoforms

UGT	Positive Control Inhibitor
UGT1A1	Bilirubin
UGT1A3	Buprenophine
UGT1A4	Hecogenin
UGT1A6	1-Naphthol
UGT1A9	Niflumic acid
UGT2B7	Diclofenac